This invention relates to a novel diagnostic method. More particularly, the invention is directed to a diagnostic method and screening test for atherosclerosis and analogous diseases involving activated phagocytes and/or inflammation such as, for example, arthritis, inflammatory bowel disease, ischemia-reperfusion injury and the like inflammatory and vascular diseases.
A leading cause of death in the United States is heart disease. About one million persons in the U.S. die of heart disease annually. Heart disease is actually a wide variety of diseases. The principal cause of many of them is atherosclerosis.
Atherosclerosis is a common form of arteriosclerosis in which fatty deposits, referred to as plaques, build up within the intima (inner wall of the arteries). As a consequence of the resulting narrowing on the arteries that feed blood to the heart, the heart's supply of oxygen, which is carried by the blood, is reduced. When blood supply is diminished appreciably, the individual may feel the pain of angina pectoris. Such pain is frequently exacerbated when the heart requires an unusually large amount of blood, e.g., during emotional stress or exercise. When the hear is thus deprived of its oxygen supply, heart muscle tissue dies. That is, a coronary occlusion or myocardial infarction may result, which can be fatal if a large area of tissue is affected.
Therefore, when an individual experiences the symptoms of angina pain, prompt contact with medical help is advised. Quick medical attention may be necessary to prevent a myocardial infarction or to provide therapeutic intervention if it has already occurred.
Although there are numerous therapeutic agents which have been used for combating heart disease, their use may be limited in instances where excessive tissue damage has already occurred. Surgical intervention such as angioplasty or bypass surgery may be indicated.
In view of the foregoing, it is apparent that a diagnostic method and screening test for atherosclerosis would have significant practical use in the medical field. Such a diagnostic would be useful for instituting precautionary measures within the individual's control, such as diet, exercise, etc., or administering therapeutic intervention before the onset of a myocardial infarction.
Accordingly, it is a principal object of the present invention to provide a diagnostic method or preventive screening test for atherosclerosis and analogous diseases involving activated phagocytes and/or inflammation such as, for example, arthritis, inflammatory bowel disease, ischemia-reperfusion injury and the like inflammatory and vascular diseases.
It is a further object to provide a diagnostic method for atherosclerosis which preferably is essentially non-invasive to the patient, as distinguished from invasive procedures such as, for example, cardiac catheterization.
(Note: Literature references on the following background information and conventional test methods and laboratory procedures well-known to the person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated in parentheses, and appended at the end of the specification.)
It is known that an increased level of low density lipoprotein (LDL) is a major risk factor for development of atherosclerotic vascular disease (1). Previous reports indicate that LDL must be oxidized to trigger the pathological events of atherosclerosis (2,3). However, the underlying pathways for oxidation in vivo have not heretofore been identified. A mechanism considered herein involves myeloperoxidase, a heme protein secreted by activated phagocytes (3,6).
Myeloperoxidase uses hydrogen peroxide (H.sub.2 O.sub.2) generated by phagocytes to generate potent microbicidal and cytotoxic agents (7-9). Catalytically active myeloperoxidase is present in human atherosclerotic lesions, where it co-localizes with lipid-laden macrophages, the cellular hallmark of the early atherosclerotic lesion (10). Patterns of immunostaining for the enzyme at different stages of atherosclerosis are remarkably similar to those for protein-bound lipid oxidation products (11).
The best characterized product of myeloperoxidase is hypochlorous acid (HOCl; refs. 5,7): EQU Cl+H.sub.2 O.sub.2 +H.sup.+ =HOCl+H.sub.2 O) (Equation 1)
This potent oxidant chlorinates electron-rich substrates and oxidatively bleaches heme groups (9,12-15). LDL exposed to reagent HOCl at neutral pH becomes aggregated and is rapidly taken up and degraded by macrophages (16).
Lipoproteins with similar properties that are rich in apolipoprotein B100, the major protein of LDL, have been isolated from atherosclerotic lesions (6,17-19). The unregulated uptake of modified LDL may be of critical importance in converting macrophages into foam cells (1-3). A monoclonal antibody which reacts selectively with HOCl-modified proteins recognizes epitopes in human atheroma and in LDL-like particles isolated from atherosclerotic tissue (6).
Myeloperoxidase is the only human enzyme known to generate HOCl at physiological concentrations of halide (7,20). However, most oxidation products generated by HOCl are either non-specific or decompose to yield uninformative compounds (9,21,22). Recent in vitro studies demonstrate that the myeloperoxidase-H.sub.2 O.sub.2 --Cl.sup.- system oxidized L-tyrosine to yield 3-chlorotyrosine (23-25).